BACKGROUND AND OBJECTIVE: DNA damage-induced chromatin reorganization is emerging to be a key aspect of eukaryotic DNA repair. Much has been learned about the role of histone modifications as landing pads for repair effectors, thereby modulating or directing their recruitment to sites of damage. Recent work suggests that structural changes in the break-surrounding chromatin environment may be equally important in directing the repair process, with implications ranging from repair factor accessibility to break-proximal transcriptional silencing. In light of this complexity, we decided to take an unbiased approach to dissect the role of chromatin in DNA repair, identify its most critical components and explore their functional relevance. RESULTS AND FUTURE DIRECTIONS: To gain insight into the role of chromatin in DSB repair, we performed RNAi-based high-throughput screening of a comprehensive list of 400 Gene Ontology-annotated chromatin modifiers. Repair efficiency was determined using the previously established, U2OS cell-based DR-GFP reporter system, in which DNA repair by homologous recombination (HR) results in restoration of a functional GFP gene and HR efficiency can, thus, be measured as the fraction of GFP+ cells. We found a large number of chromatin-modifying enzymes to be involved in DNA break repair. Specifically, Here, we identified two repressive chromatin components, the macro-histone variant macroH2A1 and the H3K9 methyltransferase and tumor suppressor PRDM2, which together modulate the choice between the antagonistic DSB repair mediators BRCA1 and 53BP1. The macroH2A1/PRDM2 module mediates an unexpected shift from accessible to condensed chromatin that requires the ATM-dependent accumulation of both proteins at DSBs to promote DSB-flanking H3K9-dimethylation. Remarkably, loss of macroH2A1 or PRDM2 as well as experimentally induced chromatin decondensation impairs BRCA1, but not 53BP1 retention at DSBs. As a result, macroH2A1 and/or PRDM2 depletion causes epistatic defects in DSB end resection, homology-directed repair and the resistance to PARP inhibition - all hallmarks of BRCA1-deficient tumors. Together, these findings identify dynamic, DSB-associated chromatin reorganization as a critical modulator of BRCA1-dependent genome maintenance. We are currently investigating the consequences of macroH2A1-associated repressive chromatin formation during replication stress, which involves HR-dependent genome maintenance pathways and has been linked to cellular dysfunction and, ultimately, senescence. Prompted by the potential of chromatin changes to selectively modulate DSB repair factor choice, we further initiated a new aim to investigate the role of other DSB repair-associated chromatin modifiers in the recruitment of BRCA1 and/or 53BP1. Based on recent reports describing ubiquitination of histone H2A as modification that is critical for 53BP1 recruitment to DSBs, we focused on a USP-family deubiquitinating enzyme that was identified as a promoter of HR in our RNAi screen. We are currently investigating how loss or overexpression of this enzyme affects the recruitment of 53BP1 and BRCA1 to sites of DSBs and concomitant DSB repair via NHEJ. We further plan to determine possible consequences of USP loss or overexpression on H2A ubiquitination. Finally, we are analyzing the impact of USP loss on genome maintenance in vivo using a previpously established mouse model. Together, this aim is expected to shed light on a novel aspect of ubiquitin-dependent DSB repair factor recruitment with the potential to distinguish between 53BP1 and BRCA1-mediated DSB repair pathways. IMPLICATIONS: Defects in BRCA1 function have been linked to tumor initiation and genomic instability. More recently, it has been suggested that a key role for BRCA1 may be to prohibit aberrant 53BP1 recruitment to sites of DNA damage, which accounts for many of the detrimental genomic effects of BRCA1 loss. Determining the factors that control the balance between BRCA1 and 53BP1 at DNA breaks is, thus, critical for our understanding of DSB repair in general and BRCA1-associated tumorigenesis in particular. Both 53BP1 and BRCA1 occupy expansive, DSB-surrounding subnuclear domains, and the identification of selective, chromatin-based modulators of BRCA1/53BP1 recruitment to sites of damage, therefore, has implications for the targeted manipulation of repair outcome and possibly tumor initiation.